Peptide potentiation of acid-sensory ion channel in pain

ABSTRACT

An assay for determining agonists, antagonists, or modulators for acid-sensing ion channels. The assay is especially useful for screening analgesics. The screening assay can be provided in a kit form. The assay comprises administering the composition to be screened to cells expressing acid-gated channels and then determining whether the composition inhibits, enhances, or has no effect on the channels when acid is introduced. The determination can be performed by analyzing whether a current is sustained by the cells in the presence of the composition and the acid. This current can be compared to that sustained by the FMRFamide and related peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. Ser. No. 09/557,506filed Apr. 25, 2000.

BACKGROUND OF THE INVENTION

[0002] FMRFamide (Phe-Met-Arg-Phe amide) and related peptides comprise afamily of neuropeptides that are abundant in many invertebrates,including Caenorhabditis elegans (Nelson, L. S., Kim, K., Memmott, J.E., and Li, C. (1998). FMRFamide-related gene family in the nematode,Caenorhabditis elegans. Mol Brain Res 58, 103-111), Aplysia californica(Greenberg, M. J., and Price, D. A. (1992). Relationships among theFMRFamide-like peptides. Prog Brain Res. 92, 25-37), and Drosophilamelanogaster (Schneider, L. E., and Taghert, P. H. (1988). Isolation andcharacterization of a Drosophila gene that encodes multipleneuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRF amide). Proc NatlAcad Sci USA 85, 1993-1997). In these organisms, FMRFamide-likeneuropeptides act as neurotransmitters and neuromodulators. At least onegene encoding FMRFamide-related peptides is present in mammals; itproduces neuropeptide FF and neuropeptide AF (A18Famide) (Perry, S. J.,Huang, E. Y. K., Cronk, D., Bagust, J., Sharma, R., Walker, R. J.,Wilson, S., and Burke, J. F. (1997). A human gene encoding morphinemodulation peptides related to NPFF and FMRF amide, FEBS Lett 409,426-430; Vilim, F. S., Aarnisalo, A. A., Nieminen, M. L., Lintunen, M.,Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P., andZiff, E. (1999). Gene for pain modulatory neuropeptide NPFF: inductionin spinal cord by noxious stimuli. Mol Pharmacol 55, 804-811). AlthoughFMRFamide itself has not been discovered in mammals (Yang, H. Y. T.,Fratta, W., Majane, E. A., and Costa, E. (1985). Isolation, sequencing,synthesis, and pharmacological characterization of two brainneuropeptides that modulate the action of morphine. Proc Natl Acad SciUSA 82, 7757-7761), administration of FMRFamide induces a variety ofphysiologic effects, including alterations in blood pressure,respiratory rate, glucose-stimulated insulin release, and behavior(Kavaliers, G. M., and Hirst, M. (1985). FMRFamide, a putativeendogenous opiate antagonist: evidence from suppression ofdefeat-induced analgesia and feeding in mice. Neuropeptides 6, 485-494;Kavaliers, M. (1987). Calcium channel blockers inhibit the antagonisticeffects of Phe-Met-Arg-Phe-amide (FMRFamide) on morphine- andstress-induced analgesia in mice. Brain Res 415, 380-384; Mues, G.,Fuchs, I., Wei, E. T., Weber, E., Evans, C. J., Barchas, J. D., andChang, J.-K. (1982). Blood pressure elevation in rats by peripheraladministration of Tyr-Gly-Gly-Phe-Met-Arg-Phe and the invertebrateneuropeptide, Phe-Met-Arg-Phe-NH2. Life Sciences 31, 2555-2561; Muthal,A. V., Mandhane, S. N., and Chopde, C. T. (1997). Central administrationof FMRFamide produces antipsychotic-like effects in rodents.Neuropeptides 31, 319-322; Nishimura, M., Ohtsuka, K., Takahashi, H.,and Yoshimura, M. (2000). Role of FMRFamide-Activated Brain SodiumChannel in Salt-Sensitive Hypertension. Hypertension 35, 443-450; Raffa,R. B., Heyman, J., and Porreca, F. (1986) Intrathecal FMRFamide(Phe-Met-Arg-Phe-NH2) induces excessive grooming behavior in mice.Neuroscience Lett 65, 94-98; Sorenson, R. L., Sasek, C. A., and Elde, R.P. (1984). Phe-Met-Arg-Phe-amide (FMRF-NH2) inhibits insulin andsomatostatin secretion and anti-FMRF-NH2 sera detects pancreaticpolypeptide cells in the rat islet. Peptides 5, 777-782; Tekegdy, G.,and Bollók, I. (1987). Amnesic action of FMRFamide in rats.Neuropeptides 10, 157-163; Thiemermann, C., Al-Damluji, S., Hecker, M.,and Vane, J. R. (1991). FMRF-amide and L-Arg-1-Phe increase bloodpressure and heart rate in the anaesthetized rate by central stimulationof the sympathetic nervous system. Biochem Biophys Res Comm 175,318-324). In mammals, FMRFamide and neuropeptide FF also modify theresponse to painful stimuli and are induced by inflammation (Kontinen,V. K., Aarnisalo, A. A., Idanpaan-Heikkila, J. J., Panula, P., andKalso, E. (1997). Neuropeptide FF in the rat spinal cord duringcarrageenan inflammation. Peptides 18, 287-292; Raffa, R. B., andConnelly, C. D. (1992). Supraspinal antinociception produced by[D-Met2]-FMRFamide in mice. Neuropeptides 22, 195-203; Tang, J., Yang,H. Y. T., and Costa, E. (1984). Inhibition of spontaneous andopiate-modified nociception by an endogenous neuropeptide withPhe-Met-Arg-Phe-NH2-like immunoreactivity. Proc Natl Acad Sci USA 81,5002-5005; Vilim, F. S., Aarnisalo, A. A., Nieminen, M. L., Lintunen,M., Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P.,and Ziff, E. (1999). Gene for pain modulatory neuropeptide NPFF:induction in spinal cord by noxious stimuli. Mol Pharmacol 55, 804-811;Yang, et al. (1985)). When FMRFamide and related peptides are injectedintracerebroventricularly, they elicit hyperalgesia and a reduction inmorphine-induced analgesia (Brussard, A. B., Kits, K. S., Ter Maat, A.,Mulder, A. H., and Schoffelmeer, A. N. M. (1989). Peripheral injectionof DNA-RFa, a FMRFa agonist, suppresses morphine-induced analgesia inrats. Peptides 10, 735-739; Kavaliers (1987); Raffa, R. B. (1988). Theaction of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides onmammals. Peptides 9, 915-922; Roumy, M., and Zajac, J. M. (1998).Neuropeptide FF, pain and analgesia. Euro J. Pharm 345, 1-11; Tang etal. (1984); Yang, et al. (1985)). In addition, FMRFamide immunoreactivematerial is released in mammals following chronic morphineadministration, and anti-FMRFamide antibodies can enhance morphine'seffects (Devillers, J. P., Boisserie, F., Laulin, J. P., Larcher, A.,and Simonnet, G. (1995). Simultaneous activation of spinal antiopioidsystem (neuropeptide FF) and pain facilitatory circuitry by stimulationof opioid receptors in rats. Brain Research 700, 173-181; Tang, et al.(1984)).

[0003] Some effects of FMRFamide and neuropeptide FF appear to bemediated through opioid receptors; these effects are blocked by theopioid antagonist naloxone (Gouarderes, C., Sutak, M., Zajak, J. M. andJhamandas, K. (1993). Antinociceptive effects of intrathecallyadministered F8Famide and FMRFamide in the rat. Eur J Pharm 237, 73-81;Kavaliers and Hirst (1985); Kavaliers (1987); Raffa (1988); Roumy andZajac (1998)). Yet other effects of FMRFamide and FMRFamide-relatedpeptides are independent of opioid receptors and are insensitive tonaloxone (Allard, M., Geoffre, S., Legendre, P., Vincent, J. D., andSimonnet, G. (1989). Characterization of rat spinal cord receptors toFLFQPQRFamide, a mammalian morphine modulating peptide: a binding study.Brain Research 500, 169-176; Gayton, R. J. (1982). Mammalian neuronalactions of FMRFamide and the structurally related opioidMet-enkephalin-Arg6-Phe7. Nature 298, 275-176; Kavaliers (1987); Raffa(1988); Raffa, et al. (1986); Roumy and Zajac (1998)). In mammals, thenon-opioid receptor(s) for FMRFamide and related peptides have not beenidentified, and it is not known how these peptides modulate painsensation. However, the discovery of a FMRFamide-activated Na⁺ channel(FaNaCh) in the mollusc Heix aspersa (Lingueglia, E., Champigny, G.,Lazdunski, M., and Barbry, P. (1995). Cloning of the amiloride-sensitiveFMRFamide peptide-gated sodium channel. Nature 378, 730-733) provided aclue that similar receptors might exist in mammals.

[0004] Unlike many neuropeptide receptors, FaNaCh is an ion channelgated directly by its peptide ligand, FMRFamide (Lingueglia, et al.(1995)). The neuropeptide receptor, FaNaCh, is a member of the DEG/ENaCfamily of channels. DEG/ENaC channels are homo- or hetero-multimerscomposed of multiple subunits (Bassilana, F., Champigny, G., Waldmann,R., de Weille, J. R., Heurteaux, C., and Lazdunski, M. (1997). Theacid-sensitive ionic channel subunit ASIC and the mammalian degenerinMDEG form a heteromultimeric H⁺-gated Na⁺ channel with novel properties.J. Biol Chem 272, 28819-28822; Coscoy, S., Lingueglia, E., Lazdunski,M., and Barbry, P. (1998). The Phe-Met-Arg-Phe-amide-activated sodiumchannel is a tetrameter. J Biol Chem 273, 8317-8322; Lingueglia, E., deWeille, J. R., Bassilana, F., Heurteaux, C., Sakai, H., Waldmann, R.,and Lazdunski, M. (1997). A modulatory subunit of acid sensing ionchannels in brain and dorsal root ganglion cells. J Biol Chem 272,29778-29783; Waldmann, R., and Lazdunski, M. (1998). H⁺-gated cationchannels: neuronal acid sensors in the NaC/DEG family of ion channels.Curr Opin Neurobiol 8, 418-424). Each subunit contains two transmembranedomains separated by a large extracellular cysteine-rich domain, andcytosolic N- and C-termini (Waldmann and Lazdunski (1998)). DEG/ENaCchannels are not voltage-gated and are cation-selective (usuallyNa⁺>K⁺). FaNaCh is the only known DEG/ENaC channel which acts as aneuropeptide receptor. Other members of this family are involved inmechanosensation, salt taste, and epithelial Na⁺ absorption (Lindemann,B. (1996). Taste reception. Physiol Rev 76, 718-766; Mano, I., andDriscoll, M. (1999). DEG/ENaC channels: a touchy superfamily thatwatches its salt. Bioessays 21, 568-578; Schild, L., Canessa, C. M.,Shimkets, R. A., Gautschi, I., Lifton, R. P., and Rossier, B. C. (1995).A mutation in the epithelial sodium channel causing Liddle diseaseincreases channel activity in the Xenopus laevis oocyte expressionsystem. Proc Natl Acad Sci USA 92, 5699-5703; Snyder, P. M., Price, M.P., McDonald, F. J., Adams, C. M., Volk, K. A., Zeiher, B. G., Stokes,J. B., and Welsh, M. J. (1995). Mechanism by which Liddle's syndromemutations increase activity of a human epithelial Na⁺ channel. Cell 83,969-978). Although a mammalian FaNaCh has not yet been isolated, mammalsdo possess multiple DEG/ENaC family members. Interestingly, one subsetof this channel family, the acid-sensing ion channels, has beenpostulated to play a role in sensory perception and may, like FMRFamide,play a role in pain perception (Waldmann and Lazdunski (1998)). Theacid-sensing DEG/ENaC channels respond to protons and generate avoltage-insensitive cation current when the extracellular solution isacidified.

[0005] The tissue acidosis associated with inflammation, infection, andischemia causes pain (Reeh, P. W., and Steen, K. H. (1996). Tissueacidosis in nociception and pain. Prog Brain Res 113, 143-151). Acidosisalso generates proton-dependent transient and sustained Na⁺ currents incultured sensory neurons (Bevan, S., and Yeats, J. (1991). Protonsactivate a cation conductance in a sub-population of rat dorsal rootganglion neurones. J Physiol (Lond) 433, 145-161; Davies, N. W., Lux, H.D., and Morad, M. (1988). Site and mechanism of activation ofproton-induced sodium current in chick dorsal root ganglion neurones. JPhysiol (Lond) 400, 159-187). Although the molecular identity of thechannels responsible for these currents is unknown, they have beenhypothesized to be acid-sensing members of the DEG/ENaC protein familybased on their ion selectivity, voltage insensitivity, and expressionpattern (Babinski, K., Le, K. T., and Seguela, P. (1999). Molecularcloning and regional distribution of a human proton receptor subunitwith biphasic functional properties. J. Neurochem 72, 51-57; Bassilana,et al. (1997); de Weille, J. R., Bassilana, F., Lazdunski, M., andWaldmann, R. (1998). Identification, functional expression andchromosomal localisation of a sustained human proton-gated cationchannel. FEBS Lett 433,257-260; Lingueglia, et al. (1997); Waldmann, R.,Bassilana, F., de Weille, J., Champigny, G., Heurteaux, C., andLazdunski, M. (1997). Molecular cloning of a non-inactivatingproton-gated Na⁺ channel specific for sensory neurons. J Biol Chem 272,20975-20978). The acid-sensing ion channels include the brain Na⁺channel 1 (BNC1) and its differentially spliced isoform MDEG2(Garcia-Anoveros, J., Derfler, B., Neville-Golden, J., Hyman, B. T., andCorey, D. P. (1997). BNaC1 and BNaC2 constitute a new family of humanneuronal sodium channels related to degenerins and epithelial sodiumchannels. Proc Natl Acad Sci USA 94, 1459-1464; Lingueglia, et al.(1997); Price, M. P., Snyder, P. M., and Welsh, M. J. (1996). Cloningand expression of a novel human brain Na⁺ channel. J Biol Chem 271,7879-7882; Waldmann, R., Champigny, G., Voilley, N., Lauritzen, I., andLazdunski, M. (1996). The mammalian degenerin MDEG, anamiloride-sensitive cation channel activated by mutations causingneurodegeneration in Caenorhabditis elegans. J Biol Chem 271,10433-10436), the acid-sensing ion channel (ASICα) and itsdifferentially spliced isoform ASICβ (Chen, C.-C., England, S., Akopian,A. N., and Wood, J. N. (1998). A sensory neuron-specific, proton-gatedion channel. Proc Natl Acad Sci USA 95, 10240-10245; Waldmann, et al.(1997)), and the dorsal root acid-sensing ion channel (DRASIC)(Mammalian neuronal DEG/ENaC channels have several names. The names ofthe three channels, listed in the order of their publication are: (1)BNC1, MDEG, BNaC1, ASIC2, and the splice variant MDEG2; (2) BNaC2,ASICα, ASIC1, and the splice variant ASICβ; and (3) DRASIC andASIC3.)(Babinski, et al. (1999); de Weille, et al. (1998); Waldmann, etal. (1997)). BNC1, MDEG2, ASICα, and DRASIC are expressed in the centralnervous system (Chen, et al. (1998); Lingueglia, et al. (1997); Olson,T. H., Riedl, M. S., Vulchanova, L., Ortiz-Gonzalez, X. R., and Elde, R.(1998). An acid sensing ion channel (ASIC) localizes to small primaryafferent neurons in rats. Neuron 9, 1109-1113; Waldmann, et al. (1997)).ASICα, ASICβ, DRASIC and MDEG2 are expressed in sensory neurons of thedorsal root ganglia (DRG) (Babinski, et al. (1999); Chen, et al. (1998);Olson, et al. (1998); Waldmann, et al. (1997)).

[0006] For the foregoing reasons, there is a need for determination andcharacterization of the roles of FMRFamide and FMRFamide-relatedpeptides in potentiation of DEG/ENaC channels, especially theacid-sensing ion channels.

SUMMARY OF THE INVENTION

[0007] The present invention identifies a family of proteins thatpotentiates the effects of a group of acid-sensing ion channels(DEG/ENaC) which are responsible for pain associated with pain fromischemia and inflammation and certain other physiological effects.

[0008] An object of the invention is an assay for screening compositionswhich effect the acid-sensing ion channels.

[0009] Another object of the invention is an assay for screeninganalgesics.

[0010] A further object of the invention is a kit which can be used forperforming the assay.

[0011] Yet another object of the invention is drug compositionsidentified by the screening assay.

[0012] These and other objects, features, and advantages will becomeapparent after review of the following description and claims of theinvention.

[0013] FMRFamide and FMRFamide-like peptides modulate acid-activatedcurrents. The present invention provides an assay for screeningcompositions to identify those which are agonists, antagonists, ormodulators of acid-sensing channels of the DEG/ENaC family. This assaycan be especially useful for determining analgesics. The assay comprisesadministering the composition to be screened to cells expressingacid-gated channels and then determining whether the compositioninhibits, enhances, or has no effect on the channels when acid isintroduced. The determination can be performed by analyzing whether acurrent is sustained by the cells in the presence of the composition andthe acid. This current can be compared to that sustained by theFMRFamide and related peptides. This assay can also be provided in kitform.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. Proton-gated currents in rat DRG neurons are modulated byFMRFamide.

[0015] (A) Trace of proton-gated whole-cell current; FMRFamide (100 μM)and pH 5 solution were present in bath during time indicated by bars.Unless otherwise indicated, pH was 7.4. N=8.

[0016] (B) Naloxone (100 μM) was present during time indicated by bar.N=3.

[0017] (C) Morphine (50 μM) and FMRFamide (50 μM) were added asindicated. N=3.

[0018] (D) Neuropeptide FF (NPFF) (50 μM) and FMRFamide (50 μM) werepresent at times indicated by bar. N=5.

[0019]FIG. 2. Effect of FMRFamide on H⁺-gated DEG/ENaC family members.Data are representative traces from Xenopus oocytes expressing ASICα(A), ASICβ (B), DRASIC (C), or BNC1 (D), from water-injected oocyte (E),and from HEK-293T cells expressing ASICα (F). Unless otherwiseindicated, extracellular pH was 7.4. FMRFamide (50 or 100 μM) and pH 5solution were present in extracellular solution during time indicated bybars. Experiments were repeated at least 7 times.

[0020]FIG. 3. FMRFamide modulates ASICα function in excised, outside-outpatches. Tracing is representative of H⁺-dependent currents recordedfrom HEK-293T cells transfected with ASICα. FMRFamide (100 μM) and pH 5solution were present in extracellular solution during time indicated bybars; otherwise pH was 7.4. N=6.

[0021]FIG. 4. Effect of order of FMRFamide and acid addition. Data arewhole-cell currents from Xenopus oocytes expressing ASICα (A, C) (n=5each), HEK-293T cells expressing ASICα (B)(n=8). Roman numerals indicatespecific interventions referred to in text. pH was 7.4 unless otherwiseindicated. FMRFamide (50 or 100 μM), and pH 5 solution were present inbath during times indicated by bars. In panel D, cell was continuouslyperfused with solution, at pH 7.4 or pH 5, for 80 sec during timeindicated by box.

[0022]FIG. 5. Properties of FMRFamide-modulated ASICα current. Data arefrom Xenopus oocytes (A, B, D-F) or HEK-293T cells (C) expressing ASICα.

[0023] (A) Effect of FMRFamide concentration on potentiation ofH⁺-dependent sustained current. Oocytes were exposed to indicatedconcentrations of FMRFamide prior to and during current activation withpH 5 solution. Measurements were normalized to the value of sustainedcurrent obtained with 500 μM FMRFamide. Data are mean±SEM; n=6-7.

[0024] (B) Effect of amiloride on FMRFamide and acid-induced sustainedcurrent. Amiloride (1 mM), FMRFamide (50 μM), and pH 5 are indicated bybars. N=5.

[0025] (C) Amiloride (100 μM), FMRFamide (100 μM), and pH 5 areindicated by bars. N=3.

[0026] (D) pH-sensitivity of ASICα current with addition of FMRFamide.FMRFamide (50 μM) was added prior to acidification. Values werenormalized to current obtained at pH 3 for the transient and theFMRFamide-modulated sustained current. Data are mean±SEM; n=7.

[0027] (E, F) Current-voltage relationships of ASICα current measured atpH 5 in the presence and absence of FMRFamide (50 μM). Extracellularbath solution containing either 116 mM Na⁺, K⁺, or Li⁺, as indicated.Membrane voltage was stepped from a holding voltage of −60 mV tovoltages of −80, −10, or +60 mV immediately before acidification.Currents from each cell were normalized to current obtained in the samecell at −80 mV in the Na⁺ solution (100%) (E) or the sustained currents(F). Data are mean±SEM; n=8 cells for Na⁺ solution and 4 cells for K⁺and Li⁺ solutions.

[0028]FIG. 6. Effect of FMRFamide-like peptides on ASICα current.Oocytes expressing ASICα were exposed to indicated peptides, morphinesulphate, or naloxone prior to and during acidification to pH 5. Allagents were tested at 50 μM and normalized to the response to FMRFamide(50 μM) obtained in the same cell, except for A18Famide (25 μM) andnaloxone (500 μM). Naloxone was applied before the addition ofFMRFamide. Data are mean±SEM for 5 to 8 cells assayed for eachcondition.

[0029]FIG. 7. Effect of FMRFamide and FRRFamide on H⁺-gated DEG/ENaCfamily members expressed in Xenopus oocytes. (A, B) ASICα and ASICβ.FMRFamide (50 μM), FRRFamide (50 μM), and pH 5 solution were present inextracellular solution during time indicated by bars. N=at least 8. (C)DRASIC. FMRFamide (100 μM), FRRFamide (100 μM), and pH 4 solution werepresent as indicated by bars. N=6.

[0030]FIG. 8. Effect of neuropeptide FF on DRASIC and ASICα expressed inXenopus oocytes. Neuropeptide FF (NPFF) (50 μM) and FMRFamide (50 μM)were present at times indicated by bars. N=5.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The current invention utilizes the finding that FMRFamide andFMRFamide-like peptides directly modulate the acid-sensing ion channels.This finding can be used to determine compositions that will be usefulin altering the response of these channels. Since these peptides andchannels appear to have a role in nociception, compositions can bescreened for inhibition of acid-sensing ion channels and antagonism ofFMRFamide-related peptides to find new analgesics. Also, sinceFMRFamide-related peptides can induce blood pressure effects, behavioreffects, and insulin and somatostatin secretion effects, screening ofcompositions with inhibiting or enhancing effects of acid-sensing ionchannels is expected to provide useful drugs which can regulate thesephysiological responses as well.

[0032] FMRFamide-related neuropeptides potentiate currents fromacid-sensing DEG/ENaC channels. The localization of acid-sensing ionchannels and FMRFamide-like peptides suggest the two may interact invivo. Both DRASIC and neuropeptide FF are found in the DRG (Allard, M.,Rousselot, P., Lombard, M. C., and Theodosis, D. T. (1999). Evidence forneuropeptide FF (FLFQRFamide) in rat dorsal root ganglia. Peptides 20,327-333; Chen, et al. (1998); Waldmann, et al. (1997)). They are alsoboth localized in the spinal cord and brain (Chen, et al. (1998);Majane, E. A., Panula, P., and Yang, H. Y. (1989). Rat brain regionaldistribution and spinal cord neuronal pathway of FLFQPQRF-NH₂, amammalian FMRF-NH₂-like peptide. Brain Res 484, 1-12; Majane, E. A., andYang, H. Y. (1987). Distribution and characterization of two putativeendogenous opiod antagonist peptides in bovine brain. Peptides 8,657-662). Moreover, FMRFamide immunoreactivity that does not appear tobe neuropeptide FF is found in DRG and the brain (Ferrarese, C.,Iadarola, M. J., Yang, H.-Y. T., and Costa, E. (1986). Peripheral andcentral origin of Phe-Met-Arg-Phe-amide immunoreactivity in rat spinalcord. Regulatory Peptides 13, 245-252; Majane and Yang (1987); Vilim, etal. (1999)). Surprisingly, FMRFamide was more potent than neuropeptideFF in activating ASIC and DRG currents.

[0033] The discovery that FMRFamide activated the molluscan FaNaChshowed that a peptide neurotransmitter could directly gate an ionchannel (Lingueglia, et al. (1995)). Several studies suggested thatFMRFamide-like peptides can activate multiple types of receptors inmammals. These may include an opiod receptor, a G protein coupledreceptor that activates second messenger pathways, and other receptorsthat so far have remained unidentified (Gherardi, N., and Zajac, J. M.(1997). Neuropeptide FF receptors of mouse olfactory bulb: bindingproperties and stimulation of adenylate cyclase activity. Peptides 18,577-583; Kavaliers (1987); Nishimura, et al. (2000); Payza, K., andYang, H. Y. (1993). Modulation of neuropeptide FF receptors by guaninenucleotides and cations in membranes of rat brain and spinal cord. JNeurochem 60, 1894-1899; Raffa and Connelly (1992)). The data of theExamples below are the first indicating that mammalian members of theDEG/ENaC channel family also respond to FMRFamide-like peptides.

[0034] Acidosis is associated with inflammation and ischemia andactivates cation channels in sensory neurons. Inflammation also inducesexpression of FMRFamide-like neuropeptides which modulate pain.Neuropeptide FF and FMRFamide generate no current on their own, butpotentiate H⁺-gated currents from cultured sensory neurons andheterologously expressed ASIC and DRASIC channels. The neuropeptidesslow inactivation and induce sustained currents during acidification.The effects are specific; different channels show distinct responses tothe various peptides. The results suggest that acid-sensing ion channelsmay integrate multiple extracellular signals to modify sensoryperception. Evidence that FMRFamide directly modulates acid-sensingchannel function includes the following:

[0035] (a) The effect of FMRFamide was not mimicked by morphine orblocked by naloxone.

[0036] (b) FMRFamide had the same effect on ASICα expressed in widelydivergent cell types, Xenopus oocytes and a human cell line. If theeffect of FMRFamide were indirect, both cell types would have to expresssimilar endogenous receptors coupled to similar second messengersystems.

[0037] (c) In cells expressing the various individual acid-gatedchannels, FMRFamide, FRRFamide, and neuropeptide FF generated currentsthat were not only quantitatively different, but, more importantly, werealso qualitatively different. If these neuropeptides had differentaffinities for an unidentified endogenous receptor coupled to a secondmessenger, then only quantitative differences would be expected.Moreover, such a scenario would predict that the quantitative effectswould be similar for the different channels. This was not the case.

[0038] (d) Application of FMRFamide altered ASICα function in excised,outside-out patches of membrane in which the cytosol is not present.

[0039] The current data show that the FMRFamide or FMRFamide-likepeptides interact with the ASIC and DRASIC channels which areevolutionarily related to the molluscan FaNaCh. However, FMRFamide didnot open these mammalian channels on its own, rather it modulated theresponse to another agonist, protons. These findings show that aFMRFamide-binding site has been at least partly conserved in theseDEG/ENaC channels, but that changes in structure have altered theconsequences of the interaction.

[0040] The alternatively spliced isoforms, ASICα and ASICβ, areidentical over most of their length; however, the amino acid sequencefrom their N-termini, through M1, and for a short distance(approximately 100 amino acids) into the extracellular domain is not thesame. Differences in the response of ASICα and ASICβ to FMRFamide andFRRFamide suggest that the more N-terminal portions of ASIC contributeto the interaction with FMRFamide. That, plus the distinct interactionsof FMRFamide and neuropeptide FF with FaNaCh and DRASIC and the lack ofa response with BNC1, provide a strategy and the reagents to investigatewhere and how these channels interact with FMRFamide and relatedpeptides.

[0041] The current data may also have implications for DEG/ENaC functionin the brain. For example, intracerebroventricular injection ofFMRFamide-related peptides induces a variety of physiologic responses(Kavaliers and Hirst (1985); Kavaliers (1987); Mues, G., Fuchs, I., Wei,E. T., Weber, E., Evans, C. J., Barchas, J. D., and Chang, J.-K. (1982).Blood pressure elevation in rats by peripheral administration ofTyr-Gly-Gly-Phe-Met-Arg-Phe and the invertebrate neuropeptide,Phe-Met-Arg-Phe-NH2. Life Sciences 31, 2555-2561; Muthal, A. V.,Mandhane, S. N., and Chopde, C. T. (1997). Central administration ofFMRFamide produces antipsychotic-like effects in rodents. Neuropeptides31, 319-322; Raffa and Connelly (1992); Raffa, et al. (1986); Roumy andZajac (1998); Sorenson, R. L., Sasek, C. A., and Elde, R. P. (1984).Phe-Met-Arg-Phe-amide (FMRF-NH2) inhibits insulin and somatostatinsecretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells inthe rat islet. Peptides 5, 777-782; Tang, et al. (1984); Thiemermann, etal. (1991); Yang, et al. (1985)). Recently, it was demonstrated that anamiloride analog inhibits FMRFamide-induced regulation of the brainrenin-angiotensin system and hypertension (Nishimura, et al. (2000)).This suggests that these channels are a target of FMRFamide in thebrain.

[0042] Proton-gated DEG/ENaC channels may function to integrate theresponse to acid and neuropeptides in the nervous system. Interestingly,another channel thought to be involved in nociception, the capsaicinreceptor, also integrates multiple stimuli, heat and acidosis (Caterina,M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., andJulius, D. (1997). The capsaicin receptor: a heat-activated ion channelin the pain pathway. Nature 389, 816-824; Tominaga, M., Caterina, M. J.,Malmberg, A. B., Rosen, T. A., Gilbert, H., Skinner, K., Raumann, B. E.,Basbaum, A. I., and Julius, D. (1998). The cloned capsaicin receptorintegrates multiple pain-producing stimuli. Neuron 21, 531-543). Thus inneurons, H⁺-gated currents could vary depending upon the type andcombinations of DEG/ENaC subunits expressed and on the presence ofdifferent FMRFamide-like neuropeptides. The diversity of channelsubunits and neuropeptides offer rich opportunities for interactions andnew targets for pharmacotherapy.

[0043] Protocols for screening new drugs, kits which utilize theprotocols, and drugs selected by the screening protocols envisioned fromthe current findings may take into account the further characterizationinformation below.

[0044] It has been suggested that tissue ischemia and inflammation causepain by stimulating H⁺-gated cation currents (Reeh and Steen (1996)).The sustained component of those currents is thought to be particularlyimportant (Bevan and Yeats (1991); Lingueglia, et al. (1997)). Thus, theability of neuropeptide FF and FMRFamide to induce sustained currentssuggests these peptides and the acid-gated channels play a role innociception. Interestingly, these peptides have been previously linkedto pain perception in the spinal cord and brain. For example, chronicinflammation induces neuropeptide FF expression in the spinal cord(Kontinen, et al. (1997); Vilim, et al. (1999)). FMRFamide-relatedpeptides may also contribute to opiate tolerance, in which increasingamounts of opiates are required to achieve the same analgesic effect(Raffa (1988); Roumy and Zajac (1998)). This may in part be explained byopiate-induced secretion of FMRFamide-related peptides from spinal cordneurons possibly inducing hypersensitivity of the nociceptive neurons(Tang, et al. (1984)).

[0045] The data indicates that the largest sustained currents in cellsexpressing ASICα required FMRFamide addition before lowering ofextracellular pH but could maintain the sustained current if the amidewas removed while the pH was being lowered. This suggests that theeffect of FMRFamide is only reversible at pH 7.4.

[0046] It is intriguing that FMRFamide should be applied before acidconsidering that ASIC and FMRFamide interact directly. The resultssuggest the following model. At pH 7.4, FMRFamide binds and is free todissociate. However, when FMRFamide is bound at pH 7.4 and then pH islowered, FMRFamide becomes trapped in the binding site. When the bindingsite is unoccupied, the channel inactivates rapidly, even in thecontinued presence of acid. However, when the binding site containsFMRFamide, channel inactivation is slowed and/or partially prevented.This scenario explains two other observations. The limited ability ofthe peptide to alter current when applied after acidification could beexplained by a conformational change at a low pH that occludes or hidesthe FMRFamide binding site (FIGS. 4A, 4B). Trapping of FMRFamide withinan occluded binding site at low pH would explain the continuedgeneration of sustained currents, even after the peptide was removedfrom the bath (FIG. 4D). This interpretation is consistent with theearlier observation that acid pH causes a conformational change in therelated BNC1 channel which altered the extracellular solventaccessibility of a specific residue (Adams, C. M., Snyder, P. M., Price,M. P., and Welsh, M. J. (1998). Protons activate brain Na⁺ channel 1 byinducing a conformational change that exposes a residue associated withneurodegeneration. J Biol Chem 273, 30204-30207).

[0047] The data from the Examples also indicates the levels of FMRFamidewhich induce changes in level of sustained currents. A level ofapproximately 1 μM induced detectable sustained currents in cellsexpressing ASICα while maximal sustained currents were achieved atapproximately 250 μM. Half-maximal sustained currents were achieved atapproximately 33 μM. The sustained current showed different cationselectivity and pH response. FMRFamide-induced sustained currents showedsensitivity to a broader range of pH compared to transient currents.

[0048] The data indicates that for the ASICα tested FMRFamide,FLRFamide, and FRRFamide were the only FMRFamide-like peptides whichcould induce sustained currents. Therefore, the channels haveneuropeptide specificity. FRRFamide showed a marked specificitydifference when tested with other channels. For ASICβ, FRRFamide slowedthe rate of inactivation without as large a sustained current asFMRFamide. For DRASIC, FRRFamide and FMRFamide increased the sustainedcurrent though at equivalent concentrations FRRFamide had a largereffect. The neuropeptide FF only had significant effects with DRASIC.

[0049] Though the details of the interactions of these peptides with thechannels is not entirely clear, the following is suggested in additionto the above information regarding specificity. N-terminal extensions ofRFamide-containing peptides did not appear to alter currents, andresults indicated that the C-terminal amide is required for a response.

[0050] Additional FMRFamide-related peptides are expected to modulateacid-gated ion channels.

[0051] The foregoing and following information indicates an assay forscreening compositions to identify those which are agonists,antagonists, or modulators of acid-sensing channels of the DEG/ENaCfamily. The assay comprises administering the composition to be screenedto cells expressing acid-gated channels in the presence of acid andFMRFamide or FMRFamide-related peptides, and determining whether thecomposition enhances or inhibits the opening of the acid-sensing ionchannels of the DEG/ENaC channel family. In addition to the ASIC andDRASIC channels, it is expected that FMRFamide or FMRFamide relatedpeptides will potentiate acid-evoked activity of other members of theDEG/ENaC cation channel family. The determination of enhancement orinhibition can be done via electrophysical analysis. Cell current can bemeasured. Alternatively, any indicator assay which detects openingand/or closing of the acid-sensing ion channels can be used such asvoltage-sensitive dyes or ion-sensitive dyes. An assay which caused celldeath in the presence of the peptide, or agonist, would be the mostdefinitive assay for indicating potentiation of the channels. Assayswhich could measure binding of FMRFamide and related peptides to thechannels could identify binding of agonists, antagonists, and modulatorsof binding. One of ordinary skill in the art would be able to determineor develop assays which would be effective in finding compositions whicheffect the acid-sensory ion channels. Since a sustained current isbelieved necessary for pain, a composition which inactivates thesustained current present when acid and FMRFamide or a related peptideactivate the acid-sensing ion channels should be useful as an analgesic.The screening can be used to determine the level of compositionnecessary by varying the level of composition administered. Thecomposition can be administered before or after addition of the acid andthe FMRFamide or a related peptide to determine whether the compositioncan be used prophylactically or as a treatment to existing pain. One ofordinary skill in the art would be able to determine other variations onthe assay(s).

[0052] Since the acid-sensing ion channels of the DEG/ENaC channelfamily and FMRFamide are implicated in other physiological responses(e.g., blood pressure, behavior, insulin and somatostatin secretion) inaddition to nociception, the assay can be used to determine compositionswhich inhibit or enhance these responses as well. For example, agonists,antagonists, or modulators of the effect of FMRFamide-related peptideson the channels could be used to alter central neuronal function toalter behavior or treat neurologic and psychiatric diseases; altermechanical sensitivity and treat conditions such as pain associated withtouch (e.g., pain associated with Herpetic neuralgia); modulate bloodpressure; alter respiration; and alter tolerance to opiods to treatopiod addiction. Additionally, these channels are potentially involvedin taste, particularly sour and salt taste. Agonists, antagonists, ormodulators of these channels and FMRFamide-related agents could be usedto inhibit or enhance specific taste sensations. Taste sensations couldbe altered in response to temperature as well since the activity ofthese channels is enhanced by cold temperature, or generally, agonistscould be used to alter the perception of cold temperature. One ofordinary skill in the art would be able to determine how to screen forthe desired effects.

[0053] Compositions which bind to the channels can be identified ordesigned (synthesized) based on the knowledge of FMRFamide potentiationof the channels and determination of the three-dimensional structure ofthe channels. These compositions could act as agonists, antagonists, ormodulators effecting nociception or other physiological responses.

EXAMPLES

[0054] Methods and Materials

[0055] cDNA Constructs

[0056] Human ASICα was cloned from brain polyA RNA. Rat ASICβ and mouseDRASIC were cloned from DRG RNA. Human BNC1 was cloned as described inPrice et al. (1996)(Price, M. P., Snyder, P. M., and Welsh, M. J.(1996). Cloning and expression of a novel human brain Na⁺ channel. JBiol Chem 271, 7879-7882). Constructs were cloned into pMT3 forexpression. The validity of the constructs was confirmed by DNAsequencing.

[0057] Cells and Expression Systems

[0058] Rat DRG neurons were cultured from Norway rats as described inBenson et al. (1999)(Benson, C. J., Eckert, S. P., and McCleskey, E. W.(1999). Acid-evoked currents in cardiac sensory neurons: A possiblemediator of myocardial ischemic sensation. Circulation Research 84,921-928). Cells were allowed to incubate overnight at room temperatureand studies were done 1 to 2 days after isolation.

[0059] Expression of the cDNA constructs in Xenopus oocytes wasaccomplished by injection of plasmid DNA into the nucleus ofdefolliculated albino Xenopus laevis oocytes (Nasco, Fort Atkinson,Wis.) as described previously (Adams, C. M., Snyder, P. M., Price, M.P., and Welsh, M. J. (1998). Protons activate brain Na⁺ channel 1 byinducing a conformational change that exposes a residue associated withneurodegeneration. J Biol Chem 273, 30204-30207). Plasmids were injectedat concentrations of 100 ng/μl for most experiments. Oocytes wereincubated in modified Barth's solution at 18° C. for 12-26 hr afterinjection. Cells injected with DRASIC were allowed to incubate for 24-48hr before analysis.

[0060] HEK-293T cells were a gift of Dr. Mark Stinski (Univ. of Iowa).ASICα cDNA was transfected into HEK-293T cells using Transfast lipidreagents (Promega, Madison, Wis.). To identify transfected cells,pGreenlantern vector encoding green fluorescent protein (Gibco,Gaithersburg, Md.) was co-transfected with ASICα at a ratio of 1:6;transfected cells were identified using epifluorescence microscopy.Cells were studied 1-2 days after transfection.

[0061] Electrophysiological Analysis

[0062] Whole-cell currents in oocytes were measured using atwo-electrode voltage-clamp as previously described (Adams, et al.(1998)). Oocytes were bathed in frog Ringers solution containing, in mM:116 NaCl, LiCl or KCl, 0.4 CaCl₂, 1 MgCl₂, 54-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4.Acidic solutions were buffered with 5 mM 2-(4-morpholino)-ethanesulfonicacid (MES) instead of HEPES. Membrane voltage was held at −60 mV unlessotherwise noted. Most peptides and naloxone were obtained from SigmaChemical Co. (St. Louis, Mo.) and were added to the extracellularsolution. The peptide FRRFamide was synthesized by Research Genetics(Huntsville, Ala.).

[0063] During whole-cell patch-clamping of DRG neurons and transfectedHEK-293T cells, the cells were bathed with an extracellular solutionthat contained, in mM: 128 NaCl, 5 MgCl₂, 1.8 CaCl₂, 5.4 KCl, 5.55glucose, 20 HEPES, pH 7.5 or 5. The pipette solution contained, in mM:120 KCl, 10 NaCl, 2 MgCl₂, 5 EGTA, 10 HEPES. Perfusion of cells withdifferent solutions was done by placing the appropriate outlet in frontof the cell. Data were recorded with an AXOPATCH 200 (Axon Instruments,Foster City, Calif.) and stored on a digital tape recorder. Digitizationwas executed by acquiring data at 400 Hz using pClamp6 (AxonInstruments, Foster City, Calif.).

[0064] Excised, outside-out patches were obtained from transfectedHEK-293T cells. The bath solution contained, in mM: 140 NaCl, 2 MgCl₂,1.8 CaCl₂, 10 HEPES at pH 7.4, or Tris(hydroxymethyl)aminomethane (Tris)or MES at pH 5. The pipette solution contained: 140 NMDG-Cl, 2 MgCl₂, 2EGTA, 10 HEPES, pH 7.4.

Example 1 FMRFamide Modulates Proton-gated Current in Rat DRG Neurons

[0065] Whole-cell patch-clamp recordings were used to investigate theeffect of FMRFamide on proton-gated currents in cultured rat DRGneurons. As previously reported (Akaike, N., and Ueno, S. (1994).Proton-induced current in neuronal cells. Prog Neurobiol 43, 73-83),acidification to pH 5 produced rapidly activating and inactivatingcurrents in the sensory neurons of the DRG (FIGS. 1A-D). FMRFamide addedalone generated no response from any of the neurons tested. However,after FMRFamide addition (50-100 μM), the inactivation ofproton-dependent current slowed, and in many neurons, there was asustained current in the continued presence of acid (FIGS. 1A and B).The presence of the neuropeptide immediately before acidification alsoaltered inactivation (FIGS. 1C, 1D).

[0066] Some effects of FMRFamide are thought to be mediated throughactivation of opiate receptors (Raffa (1988); Roumy and Zajac (1998)).To discern whether this might account for potentiation of theproton-gated currents, the effect of naloxone, an opiate antagonist, andmorphine, an opiate agonist, were used. Naloxone did not block theeffect of FMRFamide (FIG. 1B), and morphine did not mimic it (FIG. 1C).These results suggested that FMRFamide was not acting through opiodreceptors to alter current.

[0067] The mammalian FMRFamide-like neuropeptide FF(Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was also tested. Neuropeptide FFmodulated currents in a manner similar to FMRFamide; it generated nocurrent on its own, but it altered inactivation of proton-gated DRFcurrents (FIG. 1D). The effects, however, were smaller than thosegenerated by FMRFamide (FIG. 1D).

Example 2 Effect of FMRFamide on Acid-sensing Ion Channels

[0068] Members of the DEG/ENaC family are thought to be at leastpartially responsible for the acid-gated currents in the DRG. Therefore,it was reasoned that FMRFamide might have a direct effect on acid-gatedDEG/ENaC channels. Mammalian acid-sensitive ion channels in Xenopusoocytes were expressed and the resulting currents were measured. ASICαand its alternatively spliced variant ASICβ generated rapidlyinactivating currents when the extracellular pH was lowered from 7.4 to5 (FIGS. 2A, B). In contrast to its effect on FaNaCh, FMRFamide alonehad no effect on either channel. However, subsequently lowering pH inthe presence of FMRFamide potentiated the current: FIGS. 2A and 2B showslowing of inactivation and the appearance of a sustained current at pH5 in both ASICα and ASICβ. DRASIC showed a similar response in thepresence of FMRFamide (FIG. 2C); following a reduction in pH,inactivation was slowed and a sustained current was more apparent. Incontrast, the acid-gated currents from oocytes expressing BNC1 were notdiscernibly altered by FMRFamide (FIG. 2D). Neither pH nor FMRFamide inany combination produced current in control, water-injected oocytes(FIG. 2E).

[0069] FMRFamide also altered the function of ASICα expressed in thehuman cell line, HEK-293T (FIG. 2F). Acidic extracellular solutionsinduced rapidly-inactivating whole-cell currents. In the presence ofFMRFamide, inactivation slowed and a sustained current was apparent. Theeffect of FMRFamide on current from acid-gated channels expressed inXenopus oocytes and mammalian cells mimicked that observed in DRGneurons. This similarity suggested that these DEG/ENaC channels may beresponsible, at least in part, for proton-gated currents in neurons.Further studies focused on ASICα since it had been the most extensivelystudied, it is localized in nociceptive neurons of the DRG (Olson, etal. (1998)), and it produced a stable sustained current with FMRFamideaddition.

Example 3 FRFamide Modulates ASICα Current in Outside-out MembranePatches

[0070] To test whether FMRFamide interacts directly with the channel,ASICα was expressed in HEK293 cells and current from excised,outside-out patches was recorded. FIG. 3 shows that lowering theextracellular pH activated transient currents. In the presence ofFMRFamide, inactivation was slowed substantially. These data indicatedthat FMRFamide directly affects ASICα.

Example 4 Sequence of Adding FMRF Amide and Acidification

[0071] In cells expressing ASICα, the presence of FMRFamide before andduring acidification induced a sustained current (FIG. 4Aiii). Thecontinued presence of FMRFamide did not prevent channel closure when pHwas returned to 7.4 (FIG. 4Aiii). Thus, FMRFamide could neither activatenor sustain the current, rather it modulated acid-activated current.This stands in sharp contrast to FaNaCh which opens in response toFMRFamide alone and not acid (Lingueglia, et al. (1995)). The sequenceof acid and FMRFamide application was important. The largest sustainedcurrents required FMRFamide addition before lowering the extracellularpH; simultaneous addition of FMRFamide and acid (FIG. 4Avi) or additionof FMRFamide at pH 7.4 and then washing away the FMRFamide whilesimultaneously lowering pH, a sustained current still ensued (FIG. 4Av).With ASICα expressed in HEK-283T cells, the maximal sustained currentalso required addition of FMRFamide prior to acidification (FIGS. 4Biiand 4Biii); application of FMRFamide after the pH reduction failed toinduce large sustained current (FIG. 4Biv). Therefore, modulationrequired FMRFamide addition at pH 7.4 when the channel was closed.

[0072] FMRFamide could generate a sustained current, even when it wasremoved while the pH was being lowered (FIGS. 4Av, FIG. 4Biii). Similarbehavior was observed with acid-evoked currents in DRG cells (FIGS. 1Cand 1D). The effect of removing FMRFamide from the bath solution ateither pH 7.4 or pH5 was examined. FMRFamide was applied at pH 7.4, andthen the bath was continuously washed for 80 sec (FIG. 4Diii). Afterthis time, acidification generated no sustained current (FIG. 4Div).This result indicates that during the 80 sec wash, the peptidedissociated from the channel. However, when the pH was reduced whilesimultaneously removing FMRFamide, the sustained current persistedthroughout an 80 sec pH 5 wash and beyond (FIG. 4Dv). These resultssuggest that the effect of FMRFamide is only reversible at pH 7.4; oncethe channel has been activated by acid, the effect of FMRFamide isretained until the pH is returned to 7.4

Example 5 Properties of the Current Generated by pH and FMRF Amide

[0073] FMRFamide concentrations around 1 μM induced detectable sustainedcurrents in cells expressing ASICα (FIG. 5A). Maximal levels ofsustained current were achieved at ˜250 μM FMRFamide. The FMRFamideconcentration that induced half-maximal sustained currents was ˜33 μM.This concentration is higher than that reported for FaNaCh (2 μM)(Lingueglia, et al. (1995)).

[0074] Whether FMRFamide alters the properties of ASICα transientcurrents and whether the FMRFamide-generated sustained current hasproperties different from the transient current was investigated. FIGS.5B and 5C show that the FMRFamide-induced sustained current wasinhibited by amiloride in oocytes and HEK293 cells. FIG. 5D shows thatFMRFamide did not alter the pH sensitivity of the transient current. TheFMRFamide-induced sustained current, however, showed sensitivity to abroader pH range compared to the transient current. This broader rangeof sensitivity might allow a more graded pH response of theFMRFamide-bound channel. This may have implications for the perceptionof acid-evoked pain, since sustained currents are thought to play a rolein pH-dependent nociception (Bevan, S., and Geppetii, J. (1994).Protons: small stimulants of capsaicin-sensitive sensory nerves. TrendsNeurosci 17, 509-512).

[0075] The current-voltage (I-V) relationship of the H⁺-activatedtransient current of ASICα showed similar cation selectivity to what hasbeen reported previously (Waldmann, et al. (1997)); the relativepermeabilities were: Na⁺/Li⁺=0.95±0.06, and Na⁺/K⁺=6.76±0.40. The slopeconductance was similar for all the cations. The I-V relationship of thepeak current was not altered in the presence of FMRFamide (FIG. 5E). Thesustained current showed a somewhat different ion selectivity (FIG. 5F);the relative permeability was Na⁺/Li^(±)=1.05±0.07 and Na⁺/K⁺=1.25±0.2,and the slope conductivity sequence was Na⁺≧Li⁺>K⁺. The sustainedcurrent did not show Ca²⁺ conductance. Thus, FMRFamide did not alter theASICα response to pH or the properties of the initial transient current.However, the sustained current showed a different cation selectivity andpH response.

Example 6 Effect of FMRFamide-like Neuropeptides on ASICα

[0076] Since FMRFamide itself has not been found in mammals, whetherother FMRFamide-like peptides would more potently affect ASICα wasinvestigated. FMRFamide-like compounds were tested that have beenidentified in mammals including neuropeptide FF and A18Famide, whichterminate with the sequence PQRFamide (Perry, S. J., Huang, E. Y. K.,Cronk, D., Bagust, J., Sharma, R., Walker, R. J., Wilson, S., and Burke,J. F. (1997). A human gene encoding morphine modulating peptides relatedto NPFF and FMRFamide, FEBS Lett 409, 426-430; Yang, et al. (1985)) andmetenkephalin-Arg-Phe (MERF), which ends with FMRF but lacks the amide.Neither A18Famide nor MERF altered ASICα current, and neuropeptide FFproduced only minor effects on inactivation rate but no sustainedcurrent (FIG. 6 and see below). Tests were conducted of several of themany neuropeptides terminating with RFamide that have been discovered ininvertebrates (Greenberg, M. J., and Price, D. A. (1992). Relationshipsamong the FMRF-amide-like peptides. Prog Brain Res. 92, 25-37; Nelson ,L. S., Kim, K., Memmott, J. E., and Li, C. (1998). FMRFamide-relatedgene family in the nematode, Caenorhabditis elegans. Mol Brain Res 58,103-111; Perry, et al. (1997); Schneider, et al. (1988)). FLRFamide alsoinduced a sustained current in ASICα, albeit less than FMRFamide (FIG.6). N-terminal extensions of FLRFamide and other RFamide-containingpeptides identified in invertebrates did not alter ASICα currents in thepresence (FIG. 6) or absence of acid. FMRF-OH did not induce a response,indicating that the C-terminal amide is required. These results aresimilar to the neuropeptide specificity observed for FaNaCh, which hasbeen reported to only respond to FMRFamide and FLRFamide (Cottrell, G.A. (1997). The first peptide-gated ion channel. J Exp Biol 200,2377-2386). Morphine was tested to determine whether it could induce asustained current and naloxone to see if it blocked FMRFamide-inducedsustained current in Xenopus oocytes. Consistent with the results in ratDRG (FIGS. 1B and 1C), neither morphine nor naloxone altered ASICαcurrent (FIG. 6).

Example 7 Differential Effects of FMRFamide and FRRFamide

[0077] In an attempt to learn more about the peptide specificity ofacid-gated channel modulation, several FXRFamide peptides were tested.One of these, FRRFamide, showed a pronounced specificity differencebetween acid-gated channels. With ASICα, equivalent concentrations ofFRRFamide generated a sustained current similar to that produced byFMRFamide, although it was smaller in magnitude (FIG. 7A). With ASICβ,FRRFamide markedly slowed the rate of inactivation, without generatingas large a sustained current as FMRFamide (FIG. 7B). With DRASIC, bothFRRFamide and FMRFamide slowed inactivation of the transient current andincreased the sustained current, although equivalent concentrations ofFRRFamide had larger effect on transient and sustained currents (FIG.7C).

Example 8 Neuropeptide FF Potentiates DRASIC Current

[0078] Differential modulation of the various acid-sensing ion channelsby different peptides, and the finding that neuropeptide FF modulatedDRG currents, suggested that this mammalian neuropeptide should betested on all the acid-sensing channels. FIG. 8A shows that addingneuropeptide FF prior to acidification slowed the inactivation ofH⁺-gated DRASIC currents. Interestingly, the kinetics of neuropeptideFF-induced potentiation were different from those induced by FMRFamide.Neuropeptide FF had subtle effects on ASICα currents, slowinginactivation but not generating appreciable sustained current (FIG. 8B).ASICβ and BNC1 appeared unaffected by neuropeptide FF addition (data notshown).

[0079] Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those of skill in the art that it is not intended that theinvention be limited by such illustrative embodiments or mechanisms, andthat modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all such obvious modifications and variations be includedwithin the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and stepsin any sequence which is effective to meet the objectives thereintended, unless the context specifically indicates to the contrary.

1 5 1 4 PRT Caenorhabditis elegans 1 Phe Met Arg Phe 1 2 7 PRTCaenorhabditis elegans 2 Thr Gly Gly Thr Met Arg Phe 1 5 3 8 PRT Rattusnorvegicus 3 Phe Leu Phe Gln Pro Gln Arg Phe 1 5 4 6 PRT Rattusnorvegicus 4 Phe Leu Phe Gln Arg Phe 1 5 5 8 PRT Rattus norvegicus 5 PheLeu Phe Gln Pro Gln Arg Phe 1 5

What is claimed is:
 1. A method for treating conditions associated withthe response of acid-sensing ion channels of the DEG/ENaC channel familycomprising administering an agonist, antagonist, or modulator of theacid-sensing ion channels in the presence or absence of FMRFamide orFMRFamide-related peptides in a therapeutically-effective amount.
 2. Themethod of claim 1 wherein the condition is selected from the groupconsisting of pain, altered taste sensation, behavior disorder,neurologic disease, psychiatric disease, altered blood pressure, alteredrespiration, and opiod addiction.